amt of the winter mushroom, f. velutipes
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rekayasa genetikaTRANSCRIPT
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Mycobiology 34(2): 104-107 (2006)
Copyright 2006 by The Korean Society of Mycology
104
Agrobacterium-mediated Transformation of the Winter Mushroom, Flammulina
velutipes
Jung-Hee Cho, Seung-Eun Lee, Who-Bong Chang
1
and Jae-Soon Cha*
Department of Agricultural Biology, Chungbuk National University, Cheongju, Chungbuk 361-763, Korea
1
Chungbuk Agricultural Research and Extension Service, Cheongwon, Chungbuk 363-880, Korea
(Received May 16, 2006)
Flammulina velutipes was transformed efficiently by Agrobacterium-mediated transformation system. The transformation fre-
quency was about 16% with the gill tissues of the fungal fruiting body. Southern hybridization and genetic analysis suggest
that the introduced DNA was inserted onto different locations of the fungal genome, and inherited stably to the next gen-
eration via basidiospores. Transformation or gene tagging with Agrobacterium T-DNA based vector should be useful for wide
ranges of genetic or molecular biological studies of the mushroom.
KEYWORDS: Agrobacterium, Flammulina velutipes, Gene tagging, Marker inheritance, Transformation
Winter mushroom, Flammulina velutipes is widely culti-
vated in Japan (Magae et al., 2005) and Korea (Yoo et al.,
2005). This mushroom has been studied not only for prac-
tical application but also for basic research because it has
been used as a model organism of fungal graviresponse
(Kern et al., 1997; Moore and Stockus, 1998). To solve
many biological questions, molecular techniques have
been used. Among the techniques, transformation and
gene tagging are essential tools for the studies of molecu-
lar genetics, molecular breeding, and functional genomics
in economically important mushrooms. Most of transfor-
mation methods for mushrooms were based on electropo-
ration of protoplasts (Van de Rhee et al., 1996), treatment
with CaCl
2
and polyethylene glycol (Kim et al., 2003;
Ogawa et al., 1998), or restriction enzyme-mediated inte-
gration (REMI, Joh et al., 2003; Hirano et al., 2000; Irie
et al., 2003). Agrobacterium-mediated transformation
(AMT) has some advantages over the other methods. One
of them is that it does not need to prepare the protoplasts.
Since it had been reported that T-DNA from Agrobacte-
rium tumefaciens could transform Saccharomyces cerevi-
siae (Bundock et al., 1995) and some filamentous fungi
(de Groot et al., 1998), Agrobacterium-mediated transfor-
mation system was developed for many important fungi
including Agaricus bisporus (Rhee, 1996; Chen et al.,
2000; Mikosch et al., 2001). In this study, we trans-
formed economically important mushroom, Flammulina
velutipes by AMT which is the first report in F. velutipes.
Transformation of F. velutipes was carried out with the
Agrobacterium tumefaciens AGL-1 strain containing pBG-
gHg (Chen et al., 2000). The plasmid has hygromycin B
resistance gene (hph, hygromycin B phosphotranferase) as
a selection marker with the gpd (glyceraldehydes-3-phos-
phate dehydrogenase) promoter from A. bisporus and the
CaMV 35S terminator. The Agrobacterium was grown in
minimal medium (MM salts-K
2
HPO
4
2.05 g, KH
2
PO
4
1.45 g,
NaCl 0.15 g, MgSO
4
7H
2
O 0.5 g, CaCl
2
6H
2
O 0.1 g, FeSO
4
7H
2
O 2.5 mg, (NH
4
)
2
SO
4
0.5 g, and 2 g per liter; Hooy-
kaas et al., 1979) having kanamycin at 50 g/ml for 2
days at 25 using a rotary shaker set with 200 rpm. The
culture was diluted to an optical density of 0.15 at 600
nm in the induction medium (MM salts containing 40
mM MES pH 5.3, 10 mM glucose, 0.5% glycerol, and
200 M acetosyringone; Bundock et al., 1995), and fur-
ther cultured for 6 hr. The gill tissues of Flammulina
velutipes fruiting body were aseptically excised and sec-
tioned into 2 to 3-mm square pieces. The tissue pieces
were vacuum infiltrated for 10 minutes with the suspen-
sion of the bacteria grown in induction medium, and was
transferred to co-cultivation agar medium (induction medium
containing 5 mM glucose instead of 10 mM glucose).
After incubation on the co-cultivation agar for 3 days at
25
o
C~28
o
C, the tissue pieces were transferred to selection
agar medium (malt extract agar containing 50 g/ml
hygromycin, 200 M cefotaxime and 100 g/ml moxalac-
tum). The mycelium grown from the gill tissue piece on
the selection agar medium was transferred to new selec-
tion agar medium, and finally to malt extract agar
medium containing 50 g/ml hygromycin.
The hygromycin resistant transformants were cultured
in potato dextrose broth (Becton, Dickinson & Co.,
Sparks, MD, USA) for 30~45 days, and mycelial mats
were harvested from the cultures and freeze-dried. Total
DNA of the freeze dried mycelium was isolated with a
*Corresponding author
-
Agrobacterium-mediated Transformation of the Winter Mushroom, Flammulina velutipes 105
Genomic DNA Isolation Kit (NucleoGen, Siheung, Gyeo-
nggi, Korea) or with the method described by Ishii et al.
(2001). PCR was conducted using primers, gpd-FH
(5'GAAGAAGCTTTAAGAGGTCCGC3') and hph-R (5'
GGCGACCTCGTATTGGGAATC3') (Chen et al., 2000).
For Southern hybridization analysis, 10 g of total
DNA was restricted with SacI, separated on a 0.7% agar-
ose gel, and transferred to a nylon membrane by capillary
blotting. Hybridization and detection were carried out
with Dig High Prime DNA Labelling and Detection Kit II
(Roche Diagnostics GmbH, Manneheim, Germany). The
DNA amplified by PCR with gpd-FH and hph-R was
used as a probe. Fruiting body of hygromycin resistant
transformants (T0 generation) was obtained by conven-
tional cultivation method of winter mushroom. Single spore
cultures (haploid, T1 generation) were obtained from the
T0 fruiting body and maintained on PDA. Hygromycin
resistance of the T1 single spore culture was determined
on MEA containing hygromycin (50 g/ml).
It was observed that some hypha grew on the selection
agar out of the sixteen gill tissues among 100 gill tissues
treated with the A. tumefaciens AGL-1. PCR with the
primers specific for hygromycin resistance gene ampli-
fied the expected size of DNA from all 16 hygromycin
resistant transformants, but it did not form the hygromy-
cin sensitive transformants (Fig. 1). Southern hybridiza-
tion using the probe of the hygromycin resistance gene
showed the hybridized band on all hygromycin resistant
transformants (Fig. 3). These results suggest that the 16
hygromycin resistant transformants out of 100 tissues
were transformed with the marker, hygromycin resistant
gene from A. tumefaciens AGL-1. Transformation fre-
quency, 16%, is comparable to transformation frequency
of Agaricus bisporus in Chen et als study (Chen et al.,
2000) in which the gill tissue of A. bisporus was used for
transformation and the frequency was 30~40%. The sizes
of the hybridized bands detected by Southern hybridiza-
tion were different one another, which suggests the marker
genes were inserted onto different locations of the F.
velutipes genome. Kue et al. (2004) showed the hygromy-
cin resistant marker gene inserted once in different loca-
tions of F. velutipes genome by non-homologous recom-
bination in their transformation study by electroporation
of basiospores.
The 100 T1 generation single spore cultures from each
16 hygromycin resistant transformants were segregated in
their response to hyg B with the ratio of 1 resistance and
1 sensitive except (Table 1). Hygromycin resistant gene
Fig. 1. PCR analysis of DNA isolated from the hygromycin resistant transformants (T0) of F. velutipes. PCR amplification was
carried out using primers, gpd-FH and hph-R, defining a 970 bp sequence spanning the gpd promoter and the hph gene.
Lanes 2~3 for DNA from negative control and lane 4 for pBGgHg as a positive control and lanes 5~20 for DNA from
1~16 hygromycin resistant transformants.
Fig. 2. PCR analysis of DNA isolated from the single spore cultures (T1) from the hygromycin resistant transformants (T0) of F.
velutipes. PCR amplification was carried out using primers, gpd-FH and hph-R, defining a 970 bp sequence spanning the
gpd promoter and the hph gene. Lanes 2~7 for DNA from hygromycin sensitive single spore cultures and lanes 8~13 for
DNA from hygromycin resistant single spore cultures.
-
106 Cho et al.
specific DNA was amplified only in hyg
R
T1 clones (Fig.
2). The Southern hybridization also showed the hybrid-
ized band only in the hyg
R
T1 clones (data not shown).
These results indicate that the marker gene was inserted
once and the gene was maintained stably during meiosis
and inherited to the next generation via basidiospore. This
is the first result showing the transformed foreign gene
inherited to the next generation through meiosis in F.
velutipes. Kue et al. (2004) confirmed the marker gene
that is the same gene as used in this study maintained sta-
bly during mitotic cell division for 3 months in mycelium.
Transformation of F. velutipes by AMT showed in this
study is easy to carry out with moderate efficiency and
the marker gene was inserted once in the fungal genome
randomly and inherited to the next generation through
meiosis. This AMT method should be usefully applied for
the molecular genetic analysis, molecular breeding, and
biotechnological application of the economically impor-
tant winter mushroom.
Acknowledgement
This work was supported by the research grant of the
Chungbuk National University in 2004. We thank Dr. S.
Kang at The Pennsylvania State University for providing
the Agrobacterium tumefaciens strain and plasmid.
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Table 1. Segregation ratio of the hygromycin resistant
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Transformant
(T0)
Single spore cultures (T1)
2
test
Hyg
R
Hyg
S
T0-10 54 46 0.64
T0-20 50 50 0.00
T0-30 47 53 0.36
T0-40 54 46 0.64
T0-50 58 42 2.56
T0-60 59 41 3.24
T0-70 55 45 1.00
T0-80 53 47 0.36
T0-90 54 46 0.64
T0-10 60 40 4.0*
T0-11 57 43 1.96
T0-12 56 44 1.44
T0-13 50 50 0.00
T0-14 58 42 2.56
T0-15 52 48 0.16
T0-16 49 51 0.04
* Indicates significantly different (0.05 < P < 0.025).
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Agrobacterium-mediated Transformation of the Winter Mushroom, Flammulina velutipes 107
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